L-threonine is an essential amino acid and is widely used as an animal feed additive or food additive, and is also used as a raw material for medical fluid or drug synthesis. While animal protein contains a sufficient amount of L-threonine, vegetable protein is deficient in L-threonine. Thus, L-threonine is likely to be deficient in animals mainly on vegetarian diets, and thus in particular it is widely used as an additive for animal feed.
L-threonine is produced mainly by a fermentation process using Escherichia coli (E. coli) or Corynebacterium, which is developed by artificial mutagenesis or genetic recombination. To produce L-threonine, a mutant strain derived from a wild-type strain of Escherichia coli (E. coli), Corynebacteria sp., Serratia sp., or Providencia sp is used. Examples of the mutant strain include an amino acid analogue- or drug-resistant mutant strain, and a diaminopimellic acid, a methionine, a lysine, or an isoleucine auxotrophic mutant strain that has also an amino acid analogue- or drug-resistance. Among methods of producing L-threonine using a mutant strain, a method of using a microorganism that belongs to Escherichia coli species, has diaminopimellic acid and methionine auxotroph phenotypes, and is mutated so that biosynthesis of L-threonine is not affected by feedback inhibition of threonine is disclosed in Japanese Patent No. 10037/81.
A fermentation process using a recombinant strain can also be used in production of L-threonine. For example, Japanese Patent Application Publication No. 05-227977 discloses a method of producing L-threonine using a recombinant E. coli into which a threonine operon consisting of genes encoding aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase is introduced in a plasmid form, and a method of producing L-threonine using threonine-producing Brevibacterium flavum into which a threonine operon derived from E. coli is introduced (TURBA E, et al, Agric. Biol. Chem. 53:2269˜2271, 1989).
In general, the expression of a specific gene in a microorganism may be enhanced by increasing the copy number of the gene in the microorganism. For this, a plasmid that gives a greater copy number to a microorganism is used [Sambrook et al., Molecular Cloning, 2th, 1989, 1.3-1.5]. That is, the number of the gene may be increased by as many as the copy number of the plasmid per a single microorganism by inserting a target gene into the plasmid whose copy number may be maintained at a high level and then transforming the microorganism with the obtained recombinant plasmid. Attempts have also been made to enhance the productivity of threonine using this method and a partial success was reported (U.S. Pat. No. 5,538,873). However, this technology using a plasmid induces excessive expression of only a specific gene in most cases, thereby imposing a heavy burden on a host microorganism. Furthermore, plasmids may be lost during culturing of a recombinant strain, thereby decreasing plasmid stability. To address these problems of the method of producing threonine by using a recombinant strain into which a plasmid is introduced, addition of an antibiotic into a culture and methods of using a plasmid whose expression is controllable have been developed [Sambrook et al. Molecular Cloning, 2th, 1989, 1.5-1.6, 1.9-1.11]. In the case of using the plasmid whose expression is controllable, to alleviate the burden on a host microorganism and obtain a large amount of cells, during the growth phase, a host microorganism is cultured under conditions where the expression of a target gene on the plasmid does not occur, and after the sufficient growth of the host microorganism, temporary expression of the gene is induced, thereby obtaining a target material. However, methods using plasmids whose expression is controllable can be used only when a final gene product is a protein or a secondary metabolite. In a case where a gene product is a primary metabolite that is produced at the same time when microorganisms begin to grow, expression of a target gene must be induced during the growth phase. Otherwise, it is difficult to anticipate the accumulation of the primary metabolite. Since threonine belongs to a primary metabolite, the latter case is also applied to threonine.
Thus, to enhance the productivity of threonine, which is a primary metabolite, inserting genes involved in threonine biosynthesis into chromosomal DNA of a microorganism is disclosed in U.S. Pat. No. 5,939,307, instead of using a method of introducing a plasmid with threonine biosynthesis-related genes into a microorganism. Methods of increasing the threonine biosynthesis-related genes and the expression thereof have been diversely developed, but there is still a need for developing a method of more economically producing L-threonine in a high yield.
To increase the production yield of L-threonine, research on a biosynthesis pathway from oxaloacetate to threonine has been intensively conducted. With regards to this, we intended to first induce the flow of carbon along a pathway from phosphoenolpyruvate to oxaloacetate by enhancing the activity of phosphoenolpyruvate carboxylase involved in a step right before the biosynthesis of L-threonine. For this, we studied and found that a microorganism strain capable of producing L-threonine in which a promoter of a phosphoenolpyruvate carboxylase (ppc) gene on the chromosome was substituted with a promoter of a gene encoding cysteine synthase (cysK) so as to increase the expression of a gene encoding ppc, which is a first enzyme in the biosynthesis of L-threonine after glycolysis, produced L-threonine in a high yield, thus completing the present invention.